This invention pertains to amphipathic peptides having activity against bacteria and intracellular pathogens.
Bacterial resistance has hampered antibiotic therapy since the discovery of penicillin. The efficacy of current antibiotics is declining at an alarming rate due to the increase of multi-drug resistant bacteria. The lack of effectiveness arises from current antibiotics""reliance on few unique mechanisms of action. There is an unfilled need for new drugs with novel mechanisms of action upon bacterial infections.
Intracellular pathogens are especially difficult to control because they are sequestered within host cells. For example, Brucella abortus is an intracellular pathogen that lives and replicates in host macrophages. The ability to survive in macrophages allows Brucella to quickly establish a chronic infection. Treatment of chronic brucellosis is difficult because the sequestered bacteria are not exposed to the body""s immune response system: complement cascade, neutrophils, Brucella-specific antibodies, and the host""s cellular immune response. See Araya et al., xe2x80x9cTemporal Development or Protective Cell-Mediated and Humoral Immunity in BALB/c Mice Infected with Brucella abortus,xe2x80x9d Journal of Immunology, vol. 53, pp. 3330-3337 (1989).
Brucella are short, non-motile, non-sporulating, non-encapsulated, Gram-negative aerobic rods. Brucella are important veterinary pathogens. The bacteria localize in reproductive organs, mammary glands, supramammary lymph nodes, and other reticuloendothelial tissues, leading to abortion and infertility.
The zoonotic bacterial disease brucellosis has a significant impact on human health worldwide. In humans, Brucella infection results in a chronic debilitating disease known as undulant fever. Humans are exposed through direct contact with infected animals or infected animal products. In the United States, human brucellosis is an occupational hazard for veterinarians, abattoir (slaughterhouse) workers, animal handlers, and laboratory workers. Due to the highly infectious nature of the Brucella species via aerosolization, several members of the genus are candidates for biological weapons, placing military personnel at risk for infections.
Human brucellosis is characterized by malaise, fever, anorexia, muscular weakness, arthritis, and dementia. Cardiac and neurologic disorders may occur and, if untreated, may result in a mortality rate as high as 10%. Lengthy antibiotic therapy with one or multiple drugs for up to 30-45 days is required to treat brucellosis, but relapses of infection often occur after treatment is stopped. Unfortunately, antibiotic therapy does not relieve the symptoms of malaise, depression, and occasional severe dementia associated with the disease. Currently there are no vaccines available for humans; the live vaccines used in the eradication of animal brucellosis are virulent for man.
The BALB/c mouse model has been widely used for Brucella pathogenesis and vaccine efficacy studies. Inoculation of mice with virulent Brucella results in colonization of the liver and spleen. The immune response of mice to Brucella is similar to that observed in the natural hosts and humans. Cell-mediated immune (CMI) responses that aid in the clearance of infections have been defined in the murine model (Araya, et al., 1989). In general, vaccine strains that induce protective immunity in the natural hosts demonstrate abbreviated colonization profiles and also confer protection in mice.
Another example of an intracellular pathogen is Mycobacterium tuberculosis, an acid-fast Gram-positive bacterium that is the main cause of tuberculosis in humans. Tuberculosis is the leading cause of human deaths due to an infectious organism; estimates are that a third of the world""s population is currently infected. Approximately 3 million people die from tuberculosis annually, and it is expected that this number will steadily increase over the next decade as drug resistant strains proliferate.
Organisms have many defense mechanisms against invasion by pathogens, including the cellular release of defense peptides. Some defense peptides perturb the barrier function of the membrane of either the invading pathogen or of infected host cells. Although the mechanism is not completely understood, it is thought that the defense peptide forms a transmembrane channel that allows irregular ion transport across the membrane, resulting in cell lysis or death due to a loss of osmotic integrity. See Saberwal et al., xe2x80x9cCell-Lytic and Antibacterial Peptides that Act by Perturbing the Barrier Function of Membranes: Facets of their Conformational Features, Structure-Function Correlations and Membrane-Perturbing Abilities,xe2x80x9d Biochimica et Biophysica Acta, vol. 1197, pp. 109-131 (1994).
Many antimicrobial peptides selectively inhibit and kill bacterial cells while maintaining low cytotoxicity for normal mammalian cells. The selectivity for pathogens has been attributed to a difference between bacterial and mammalian cell membranes. The exterior membranes of bacteria are negatively charged, whereas mammalian cell exterior membranes are generally neutral. Antimicrobial peptides are positively charged and therefore may preferentially bind to bacterial membranes. The cholesterol in mammalian cell membranes has also been suggested as the basis for the selectivity of antimicrobial peptides. See Maloy et al., xe2x80x9cStructure-Activity Studies on Magainins and Other Host Defense Peptides,xe2x80x9d Biopolymers (Peptide Science), vol. 37, 105-122 (1995). Membrane disruption by the antimicrobial peptides could be inhibited by cholesterol. Finally the lower membrane potential across mammalian cells, or some combination of the above factors, could be responsible for the observed selectivity of the antimicrobial peptides between bacteria and normal mammalian cells.
The specificity of various antimicrobial peptides differs. For example, melittin, a component of honeybee venom, is not selective. The minimum bactericidal concentration of melittin also damages normal mammalian cells. By contrast, the naturally occurring magainins and cecropins exhibit substantial bactericidal activity at concentrations that are not lethal to normal mammalian cells. It has been found that sequence homology is not a prerequisite for biological activity. (Saberwal et al., 1994) Many natural antimicrobial peptides of widely varying sequences have been isolated. One consistent structural feature is the presence of an amphipathic helical domain. Synthetic analogs of native peptides with amino acid-substitutions expected to enhance amphipathicity and helicity have shown increased biological activity. However, most analogs with increased antimicrobial activity unfortunately also show increased cytotoxicity against normal mammalian cells. Notable exceptions are the melittin-cecropin hybrids, which are more bacteriostatic than cecropins and less cytotoxic than melittin. Melittin, a 26-residue peptide, is cytotoxic and has broad spectrum antimicrobial activity at micromolar concentrations. There are a number of other natural amphipathic peptides that are much less cytotoxic than melittin, but that have comparable broad spectrum antimicrobial activity. Magainins and cecropins exhibit bacteriostatic and bactericidal activity at concentrations that are not cytotoxic toward normal mammalian cells. These peptides are unstructured in dilute aqueous solution, but become helical in amphipathic media such as micelles, synthetic bilayers, and cell membranes.
Some defense peptides have been reported to selectively attack host cells infected with an intracellular pathogen, while not affecting normal mammalian cells. See Barr et al., xe2x80x9cActivity of Lytic Peptides Against Intracellular Trypanosoma cruzi Amastigotes in vitro and Parasitemias in Mice,xe2x80x9d Journal of Parasitology, vol. 81, no. 6, pp. 974-978 (1995). Such selectivity has not been previously reported for Brucella infection. There have been no previous studies on in vivo activity of a peptide against an intracellular pathogen. The mechanism for peptide selectivity against host cells infected with a pathogen is unknown. Three possibilities include the following: (1) the peptide has greater binding affinity to infected host cells; (2) the peptide binds to both infected and normal cells, but the normal cell is able to repair the membrane; and (3) the membrane of the infected cell is inherently defective, so the binding of the peptide causes cell death. See Jaynes et al., xe2x80x9cIn vitro Cytocidal Effect of Lytic Peptides in Several Transformed Mammalian Cell Lines,xe2x80x9d Peptide Research, vol. 2, pp. 157-160 (1989).
Antimicrobial peptides generally have random coil conformations in dilute aqueous solutions. However, high levels of helicity can be induced by helix-promoting organic solvents and amphipathic media such as micelles, synthetic bilayers, and cell membranes. When helicity is induced, the polar and nonpolar amino acid residues are aligned into an amphipathic helix. The amphipathic xcex1-helix is a common structural motif of many proteins and biologically active peptides. An amphipathic peptide or protein is one in which the hydrophobic amino acid residues are predominantly on one side of the helix looking down the helical axis, while the hydrophilic amino acid residues are predominantly on the opposite side, resulting in a peptide or protein that is predominantly hydrophobic on one face, and predominantly hydrophilic on the opposite face. Amphipathic helical domains are found in membrane recognition sites, such as specific ion channel proteins, signal peptides, and antimicrobial and venom peptides. The interaction of amphipathic peptides with membranes depends at least in part on the relative sizes of the hydrophobic and hydrophilic faces and the charge density of the hydrophilic face. Natural antimicrobial peptides generally have an equivalent number of polar and nonpolar residues within the amphipathic domains, and enough basic residues to give the peptide an overall positive charge at neutral pH.
Antimicrobial peptides based upon an amphipathic xcex1-helix have been known for decades (Saberwal et al., 1994; Epand et al., xe2x80x9cMechanisms for the Modulation of Membrane Bilayer Properties by Amphipathic Helical Peptides,xe2x80x9d Biopolymers (Peptide Science) vol. 37, pp. 319-338 (1995)). The mechanism of action of this class of peptides, typified by cecropins from the Hyalophora moth and magainins from the African clawed frog, Xenopus laevis, is still a matter of controversy (Epand, et al., 1995). There is abundant evidence that these peptides disrupt the barrier function of the membranes of susceptible cells.
Many natural peptides of this class have now been isolated from taxa ranging from bacteria to mammals. De novo peptides having xcex1-helical domains likely to form amphipathic xcex1-helices have been synthesized and shown to have in vitro antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria. See Javadpour et al., xe2x80x9cDe Novo Antimicrobial Peptides with Low Mammalian Cell Toxicity,xe2x80x9d Journal of Medicinal Chemistry, vol. 39, pp. 3107-3113 (1996). Several examples demonstrate that chiral recognition is not required, as all L-peptides or all D-peptides have the same bioactivity in vitro. In addition to having an amphipathic xcex1-helix, there is apparently a need for at least some overall positive charge for the peptide to be active. Essentially all the natural peptides have at least one overall positive charge, but these peptides also usually have some uncharged polar and even some negatively charged amino acids that help to form the polar face of the amphipathic domain.
Careful studies of the structure and orientation of the peptides show that peptides of this class bind as xcex1-helices, with the nonpolar face of the peptides embedded in the phospholipid membrane, lying perpendicular to the fatty acid chains of the phospholipids (Epand et al., 1995). This conformation is called xe2x80x9cpeptide raftingxe2x80x9d since the hydrophobic faces of the peptides partially sink into the phospholipid surface and may become loosely bound together, like the logs of a raft, causing cell membrane disruption. An alternative explanation is that while the vast majority of peptides lie perpendicular to phospholipid bilayer, one or a few peptide aggregates could self-assemble as nonspecific transmembrane pores. (Epand et al., 1995). A transmembrane channel model suggests a minimum xcex1-helix length would be required to span the hydrophobic core of the phospholipid bilayer. The average hydrophobic core of a bilayer at equilibrium is about 40 xc3x85. Spanning this length would require a peptide of about 20 residues.
Synthetic analogs of several naturally occurring cytotoxic peptides have previously been synthesized, but in the past it has been laborious and expensive to synthesize these synthetic peptides in large quantities. There is a continuing need for synthetic antimicrobial peptides that are easy to synthesize, and that exhibit antibacterial activity or activity against intracellular pathogens at concentrations that are not lethal to normal mammalian cells.
One of the earliest designed peptides was a melittin analog with a simplified N-terminus and the native C-terminal segment that had hemolytic activity comparable to that of melittin. Another designed amphipathic xcex1-helical peptide had 12 to 22 residues, composed of 2:1 Leu and Lys residues, with a narrow polar face. The peptides with 15, 20 and 22 residues showed over 10-fold higher hemolytic activity than melittin. See Cornut et al., xe2x80x9cThe Amphipathic xcex1-Helix Concept: Application to the De Novo Design of Ideally Amphipathic Leu, Lys Peptides with Hemolytic Activity Higher than That of Melittin,xe2x80x9d FEBS (Federation of European Biochemical Societies) Letters, vol. 349, pp. 29-33 (1994). D-Melittin, D-magainin, and D-cecropin derivatives have biological activities that are essentially the same as those of the native L-peptides. De novo peptides that use the amphipathic helix as a starting point have been synthesized and have exhibited bacteriostatic and cytotoxic activities similar to those of the native peptides. See Lee et al., xe2x80x9cRelationship Between Antimicrobial Activity and Amphipathic Property of Basic Model Peptides,xe2x80x9d Biochimica et Biophysica Acta, vol. 862, pp. 211-219 (1986); and Blondelle et al., xe2x80x9cDesign of Model Amphipathic Peptides Having Potent Antimicrobial Activities, Biochemistry, vol. 31, pp. 12688-12694 (1992). Structure-activity studies have shown a relationship between antibacterial activity, peptide length, and the proportion of positively-charged and hydrophobic residues. See Bessalle et al., xe2x80x9cStructure-Function Studies of Amphiphilic Antibacterial Peptides,xe2x80x9d Journal of Medicinal Chemistry, vol. 36, pp. 1203-1209 (1993).
One class of natural antimicrobial peptides, peptaibols, have several xcex1-aminoisobutyric acid (Aib) residues, are acetylated on the N-terminus, and have an amino alcohol at the C-terminus. See Benedetti et al., xe2x80x9cPeptaibol Antibiotics: A Study on the Helical Structure of the 2-9 Sequence of Emerimicins III and IV,xe2x80x9d Proceedings of the National Academy of Sciences, vol. 79, pp. 7951-7954 (1982). The Aib residue is a nonpolar xcex1,xcex1-dialkylated amino acid (an xcex1-amino acid residue that is dialkylated at the xcex1 carbon), which is believed to stabilize a helical structure. For example, alamethicin, a 20-residue peptide, is rich in Aib residues (up to 50%) and exists primarily as either an (xcex1- or 310-helix. (In an xcex1 helix the NH of the i+4 residue is hydrogen bonded to the CO of residue i (5xe2x86x921), whereas in the 310 helix the i+3 NH group participates in a 4xe2x86x921 hydrogen bond.) This peptide is known to form membrane channels. See Nagaraj et al., xe2x80x9cAlamethicin, a Transmembrane Channel,xe2x80x9d Accounts of Chemical Research, vol. 14, pp. 356-362 (1981). In addition, peptides with several xcex1,xcex1-dialkylated amino acids throughout the sequence are resistant to enzymatic hydrolysis, especially by trypsin, which enhances in vivo activity.
The nonpolar xcex1,xcex1-dialkylated amino acids (or hydrophobic xcex1,xcex1-dialkylated amino acids), especially Aib, have been incorporated into peptides to control the secondary structure of the peptide. See Karle et al., xe2x80x9cStructural Characteristics of xcex1-Helical Peptide Molecules Containing Aib Residues,xe2x80x9d Biochemistry, vol. 29, no. 29, pp. 6747-6756 (1990).
One polar xcex1,xcex1-dialkylated amino acid (i.e., a substituted, hydrophilic xcex1,xcex1-dialkylated amino acid) that has been synthesized is 4-aminopiperidine-4-carboxylic acid (Api). See Jacobsen et al., xe2x80x9cPotential GABA Uptake Inhibitors. Synthesis and Relative Stereochemistry of Some Aminopiperidinecarboxylic acids,xe2x80x9d Acta Chemica Scandinavica B, vol. 34, pp. 319-326 (1980). There is no known prior report incorporating a polar xcex1,xcex1-dialkylated amino acid into a peptide. There is no known prior report of the preparation of a protected derivative of a polar xcex1,xcex1-dialkylated amino acid that would be suitable for peptide synthesis.
Peptides with xcex1,xcex1-dialkylated amino acids are sterically constrained because of the disubstitution about the xcex1-carbon. There are two potential helical conformations for peptides containing xcex1,xcex1-dialkylated amino acids: 310 or xcex1-helical. In larger peptides, increasing the number of xcex1,xcex1-dialkylated amino acids tends to promote specific helical conformation. The more xcex1,xcex1-dialkylated amino acids that are used in a peptide the greater the steric constraint toward helicity. For Aib containing peptides, it appears that 50% xcex1,xcex1-dialkylated amino acids induce 310-helical conformation in peptides of 10 residues. See Basu et al., xe2x80x9cConformational Preferences of Oligopeptides Rich in xcex1-Aminoisobutyric Acid. I. Observation of a 310/xcex1-Helical Transition upon Sequence Permutation,xe2x80x9d Biopolymers, vol. 31, pp. 1763-1774 (1991). Additionally, 24-residue peptides with a lysine or arginine at every third residue have been reported to be an amphipathic 310-helix. However, this peptide had no antimicrobial activity. See Iwata et al., xe2x80x9cDesign and Synthesis of Amphipathic 310-Helical Peptides and Their Interactions with Phospholipid Bilayers and Ion Channel Formation,xe2x80x9d Journal of Biological Chemistry, vol. 269, no. 7, pp. 4928-4933 (1994).
The overall length of the peptide plays a role in determining the conformation, in conjunction with the number of xcex1,xcex1-dialkylated amino acids. The effect of a single xcex1,xcex1-dialkylated amino acid in influencing the secondary structure is greatest in small peptides; as the length of peptide chain increases the effect diminishes. Thus it is necessary to increase the percentage of xcex1,xcex1-dialkylated amino acids in order to promote the 310 helical conformation (Bendetti et al., 1982).
S. E. Blondelle et al., xe2x80x9cDesign of Model Amphipathic Peptides Having Potent Antimicrobial Activities,xe2x80x9d Biochemistry, vol. 31, pp. 12688-12694 (1992) reports that the sequence LKLLKKLLKKLKKLLKKL (SEQ ID NO. 10) adopts an xcex1-helical conformation, and that the peptide has activity against both Gram-positive and Gram-negative bacteria. The activities of certain analogs having various substitutions, omissions, and lengths are also discussed.
W. F. DeGrado et al., xe2x80x9cConformationally Constrained xcex1-Helical Peptide Models for Protein Ion Channels,xe2x80x9d Biopolymers, vol. 29, pp. 205-213 (1990) discusses amphiphilic xcex1-helical models for ion channels formed from various peptides composed of only Leu and Ser residues, or a modification of such a peptide incorporating xcex1-aminoisobutyric acid.
We have invented novel xe2x80x9cminimalistxe2x80x9d antimicrobial peptides, based on 50 to 80% xcex1,xcex1-dialkylated amino acids, that may be readily synthesized on a large scale, for example by standard Fmoc solid-phase synthesis techniques. The novel peptides are short, cationic, amphipathic, and form a helix. We have also invented new polar xcex1,xcex1-dialkylated amino acids and a process to produce protected derivatives of polar xcex1,xcex1-dialkylated amino acids for incorporation into peptides. These synthetic peptides are easy and inexpensive to synthesize via solid-phase techniques. The present invention has found peptides as short as 10 residues that exhibit antibacterial activity at concentrations that are not lethal to normal mammalian cells, and that also exhibit bioactivity against intracellular pathogens. These synthetic peptides with a high percentage of xcex1,xcex1-dialkylated amino acids were also found to be resistant to tryptic digestion.